Ballistic transfection with dendrimers

ABSTRACT

The present invention relates to compositions and methods involving biocompatible dendrimers. In particular, the present invention provides dendrimeric copolymers with poly(propyleneimine) (POPAM) interiors and poly(amidoamine) (PAMAM) exteriors for use in transfection and imaging applications.

[0001] This invention was made in part with Government support by theUnited States Army Research Laboratory Grant Number DAAL01-96-2-0044, bythe National Cancer Institute Grant Number NOI-CO-97111, by the NationalInstitutes of Health Grant Number N01-AR-6-2226, and by the DefenseAdvanced Research Projects Agency Grant Number MDA972-97-1-0007.Accordingly, the Government has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methodsinvolving biocompatible dendrimers. In particular, the present inventionprovides dendrimeric copolymers with poly(propyleneimine) (POPAM)interiors and poly(amidoamine) (PAMAM) exteriors for use in transfectionand imaging applications.

BACKGROUND OF THE INVENTION

[0003] Dendrimers and hyperbranched polymers represent a novel class ofstructurally controlled macromolecules derived from abranches-upon-branches structural motif (Tomalia et al., Angew. Chem.Intl. Edit. 29:138-175 [1990]; and Naylor et al., J. Am. Chem. Soc.111:2339-2341 [1989]). Dendrimers are well defined, highly-branchedmacromolecules that radiate from a simple organic molecule as a core andare synthesized through a stepwise, repetitive reaction sequence thatguarantees complete shells for each generation leading theoretically toproducts that are unimolecular and monodisperse (Tomalia et al.,Macromolecule 24:1435-1438 [1999]; and Dvornic and Tomalia, “Dendriticpolymers divergent synthesis: starburst poly(amidoamine) dendrimers,” inSalamone (ed.) The Polymeric Materials Encyclopedia: Synthesis,Properties and Applications,” (CRC Press: Boca Raton) [1996]). Thesynthetic procedures developed for dendrimer preparation permit nearlycomplete control over the critical molecular design parameters, such assize, shape and shell/core chemistry. Synthetic techniques that haveproven effective for dendrimer production include the divergent strategyof Tomalia and co-workers (Tomalia et al., Angew. Chem. Intl. Edit.29:138-175 [1990]; and Naylor et al., J. Am. Chem. Soc. 111:2339-2341[1989]), the convergent growth strategy of Fréchet and co-workers(Hawker et al., J. Chem. Soc. Perkins Trans. 12:1287-1297 [1993];Fréchet, Science 263:1710-1715 [1994]; and Fréchet, Science269:1080-1083 [1995]), and the self-assembly strategy of Zimmerman andco-workers (Zimmerman et al., Science 271:1095-1098 [1996]). Thesemethods have made possible the generation of synthetic macromoleculeswith unique combinations of properties (Bell, Science 271: 1077-1078;van Hest et al., Science 268:1592-1595 [1995]; Jansen et al., J. Am.Chem. Soc. 117:4417-4418 [1995]; and Jansen et al., Science266:1226-1229 [1995]).

SUMMARY OF THE INVENTION

[0004] The present invention relates to compositions and methodsinvolving biocompatible dendrimers. In particular, the present inventionprovides dendrimeric copolymers with poly(propyleneimine) (POPAM)interiors and poly(amidoamine) (PAMAM) exteriors for use in transfectionand imaging applications.

[0005] For example, the present invention provides a compositioncomprising a hybrid dendrimer having a poly(propyleneimine) interior anda poly(amidoamine) exterior. In some embodiments, thepoly(propyleneimine) interior is a dendrimer selected from the groupconsisting of a generation 2 dendrimer with sixteen amine surfacegroups, a generation 3 dendrimer with 32 amine surface groups, and ageneration 4 dendrimer with 64 amine surface groups, although otherdendrimers may be used. In some embodiments, the poly(amidoamine)exterior comprises one or more shells (e.g., 1, 2, 3, 4, 5, etc.). Insome preferred embodiments, the hybrid dendrimer has a 1,4-diaminobutanecore.

[0006] In some embodiments, the hybrid dendrimer further comprising aguest molecule. The present invention is not limited by the nature ofthe guest molecule. A number of exemplary guest molecules are disclosedherein. In some preferred embodiments, the guest molecule comprises anucleic acid molecule, a metal, and/or a drug.

[0007] The present invention also provides methods for preparing ahybrid dendrimer comprising: providing an amine-terminatedpoly(propyleneimine) dendrimer, methyl acrylate, and ethylenediamine;reacting said amine-terminated poly(propyleneimine) dendrimer with saidmethyl acrylate to produce an ester-terminated compound; and reactingsaid ester-terminated compound with ethylenediamine to produce saidhybrid dendrimer. In some embodiments, the method further comprises thestep of d) attaching a guest molecule to said hybrid dendrimer. In someembodiments, the amine-terminated poly(propyleneimine) dendrimercomprises a guest molecule (e.g., which gets incorporated into thehybrid dendrimer when the out layers are added). In some preferredembodiments, the reacting steps are conducted in a methanol solventunder an intert nitrogen atmosphere. The present invention providescompositions comprising the hybrid dendrimer prepared according to suchmethods. In some embodiments, the hybrid dendrimer has a hydrodynamicdiameter of from 10 to 100 angstroms.

[0008] The present invention also provides methods for transfectingcells comprising: providing dendrimer/nucleic acid complexes and targetcells; and ballistically accelerating the dendrimer/nucleic acidcomplexes at the target cells under conditions such that nucleic acidenters the target cells. In some embodiments, the dendrimer/nucleic acidcomplexes comprise PAMAM dendrimers. In some embodiments, thedendrimer/nucleic acid complexes comprise hybrid dendrimers having apoly(propyleneimine) interior and a poly(amidoamine) exterior. In somepreferred embodiments, the dendrimer/nucleic acid complexes comprisemetal particles (e.g., gold and/or silver particles). In some preferredembodiments, the dendrimer/nucleic acid complexes have a charge ratio of1 or less (e.g., 0.1 or less). In some preferred embodiments, thedendrimer/nucleic acid complexes comprise 99% or greater monodispersedparticles. In some embodiments, the ballistically accelerating step iscarried out by a ballistic device. In some preferred embodiments, theballistic device is held less than one centimeter from said target cellsduring said ballistically accelerating step.

DESCRIPTION OF THE FIGURES

[0009] The following figures form part of the specification and areincluded to further demonstrate certain aspects and embodiments of thepresent invention. The invention may be better understood by referenceto one or more of these figures in combination with the detaileddescription of specific embodiments presented herein.

[0010]FIG. 1 shows POPAM and PAMAM terminal branch compositions in panelA, and the composition of a POMAM dendrimeric copolymer in panel B.

[0011]FIG. 2 shows a schematic of the divergent, reiterative, two-stepPAMAM dendronization reactions of the POMAM dendrimeric copolymer. Thefirst step involves Michael addition of methylacrylate (acrylate esters)to the terminal amine groups. The second step involves amidation withethylenediamine.

[0012]FIG. 3 shows a representative ¹³C NMR spectrum for a generation2:1 POMAM hybrid dendrimer.

[0013]FIG. 4 shows that the experimental (panel A) and theoretical(panel B) molecular weights of the POMAM hybrid dendrimers are dependentupon both the generation number and the number of shells.

[0014]FIG. 5, panel A shows the GPC eluogram of a generation 2:2 POMAMhybrid dendrimer. Panel B shows a GPC eluogram comparison of POMAMhybrid dendrimers of the generations 0 to 7.

[0015]FIG. 6 shows that the hydrodynamic diameter of the POMAM hybriddendrimers is a function of the generation number and the number ofshells.

[0016]FIG. 7, panel A shows HPLC eluograms of generation 2:4, 3:4, and4:4, POMAM hybrid dendrimers. Panel B shows a plot of the retention timeversus logarithm of the molecular weight of PAMAM and POMAM hybriddendrimers.

[0017]FIG. 8 shows a tapping mode AFM image of generation 4:2 POMAMhybrid dendrimer molecules. The sample was prepared by spreadingapproximately one drop of a 1×10⁻⁴ percent (weight) solution on afreshly cleaved mica surface by spin coating and drying at roomtemperature.

[0018]FIG. 9 shows the potentiometric tritration curve of the generation2:2 POMAM hybrid dendrimer.

[0019]FIG. 10 shows the activity of the CAT transgene expressed bymurine skin 48 hrs after in vivo ballistic delivery of generation 5PAMAM dendrimer/DNA complexes. An orifice-to-target distance of 0 or 1.5cm was used. The formulations of the samples are indicated along thex-axis, with the dendrimer/DNA charge ratio shown in parentheses.

[0020]FIG. 11 shows the activity of the CAT transgene expressed by humanskin grafts 24 hrs after ballistic delivery of dendrimer/DNA complexes.Human skin grafts established on the dorsal side of SCID mice weretransfected with various dendrimer/DNA formulations as indicated alongthe x-axis.

[0021]FIG. 12 shows TEM images of human skin grafts after ballisticdelivery of Ag nanocomposite dendrimer/DNA complexes. Electron-denseparticles localized in the cell nucleus (panel A), in the nucleus and inthe cytoplasm (panel B) and in the acellular matrix (panel C). Themagnification is approximately 4000×.

[0022]FIG. 13 shows the dose-dependent efficiency of dendrimer-mediatedballistic transfection of murine skin in vivo. Approximately 10 or 50 μgof reporter plasmid DNA was used alone or complexed with a generation 5PAMAM dendrimer at 0.01 charge ratio. The CAT activity was measured 48hr after transfection.

[0023]FIG. 14 shows the efficiency and duration of transgene expressionfollowing ballistic transfection of murine skin in vivo. BALB/c miceskin was transfected with the indicated amounts of either DNA alone ordendrimer/DNA complexes at 0.05 charge ratio. The CAT activity wasmeasured 48 hr and 7 days after transfection.

DEFINITIONS

[0024] To facilitate an understanding of the present invention, a numberof terms and phrases are defined below:

[0025] As used herein, the terms “biocompatible” and “biofriendly” referto compositions comprised of natural or synthetic materials, in anysuitable combination, that remain substantially biologically unreactivein a host. The term “substantially unreactive” means that any responseobserved in a host is a subclinical response (e.g., a response that doesnot necessitate therapy). The term “gene” refers to a nucleic acid(e.g., DNA) sequence that comprises coding sequences necessary for theproduction of a polypeptide or precursor. The polypeptide can be encodedby a full length coding sequence or by any portion of the codingsequence so long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, etc.) of thefull-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the including sequences locatedadjacent to the coding region on both the 5′ and 3′ ends for a distanceof about 1 kb or more on either end such that the gene corresponds tothe length of the full-length mRNA. The sequences that are located 5′ ofthe coding region and which are present on the mRNA are referred to as5′ non-translated sequences. The sequences that are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene which are transcribed into nuclear RNA (hnRNA);introns may contain regulatory elements such as enhancers. Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide.

[0026] As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

[0027] As used herein, the term “antisense” is used in reference to DNAor RNA sequences that are complementary to a specific DNA or RNAsequence (e.g., mRNA). Included within this definition are antisense RNA(“asRNA”) molecules involved in gene regulation by bacteria. AntisenseRNA may be produced by any method, including synthesis by splicing thegene(s) of interest in a reverse orientation to a viral promoter whichpermits the synthesis of a coding strand. Once introduced into anembryo, this transcribed strand combines with natural mRNA produced bythe embryo to form duplexes. These duplexes then block either thefurther transcription of the mRNA or its translation. In this manner,mutant phenotypes may be generated. The term “antisense strand” is usedin reference to a nucleic acid strand that is complementary to the“sense” strand. The designation (−) (i.e., “negative”) is sometimes usedin reference to the antisense strand, with the designation (+) sometimesused in reference to the sense (i.e., “positive”) strand.

[0028] Where amino acid sequence is recited herein to refer to an aminoacid sequence of a naturally occurring protein molecule, amino acidsequence and like terms, such as polypeptide or protein are not meant tolimit the amino acid sequence to the complete, native amino acidsequence associated with the recited protein molecule.

[0029] The term “transgene” as used herein refers to a foreign gene thatis placed into an organism. The term “foreign gene” refers to anynucleic acid (e.g., gene sequence) that is introduced into the genome ofan animal by experimental manipulations and may include gene sequencesfound in that animal so long as the introduced gene does not reside inthe same location as does the naturally-occurring gene.

[0030] As used herein, the term “vector” is used in reference to nucleicacid molecules that transfer DNA segment(s) from one cell to another.The term “vehicle” is sometimes used interchangeably with “vector.”Vectors are often derived from plasmids, bacteriophages, or plant oranimal viruses.

[0031] The term “expression vector” as used herein refers to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences necessary for expression in prokaryotes usually include apromoter, an operator (optional), and a ribosome binding site, oftenalong with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

[0032] The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher than that typically observedin a given tissue in a control or non-transgenic animal. Levels of mRNAare measured using any of a number of techniques known to those skilledin the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the RAD50mRNA-specific signal observed on Northern blots).

[0033] As used herein, the term “gene transfer system” refers to anymeans of delivering a composition comprising a nucleic acid sequence toa cell or tissue. For example, gene transfer systems include, but arenot limited to vectors (e.g., retroviral, adenoviral, adeno-associatedviral, and other nucleic acid-based delivery systems), microinjection ofnaked nucleic acid, dendrimers, and polymer-based delivery systems(e.g., liposome-based and metallic particle-based systems). As usedherein, the term “viral gene transfer system” refers to gene transfersystems comprising viral elements (e.g., intact viruses and modifiedviruses) to facilitate delivery of the sample to a desired cell ortissue.

[0034] The term “transfection” as used herein refers to the introductionof foreign DNA into eukaryotic cells. Transfection may be accomplishedby a variety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, biolistics, anddendrimers.

[0035] The term “stable transfection” or “stably transfected” refers tothe introduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

[0036] The term “transient transfection” or “transiently transfected”refers to the introduction of foreign DNA into a cell where the foreignDNA fails to integrate into the genome of the transfected cell. Theforeign DNA persists in the nucleus of the transfected cell for severaldays. During this time the foreign DNA is subject to the regulatorycontrols that govern the expression of endogenous genes in thechromosomes. The term “transient transfectant” refers to cells that havetaken up foreign DNA but have failed to integrate this DNA.

[0037] As used herein, the term “recombinant DNA molecule” as usedherein refers to a DNA molecule that is comprised of segments of DNAjoined together by means of molecular biological techniques.

[0038] The term “test compound” refers to any chemical entity,pharmaceutical, drug, and the like that can be used to treat or preventa disease, illness, sickness, or disorder of bodily function. Testcompounds comprise both known and potential therapeutic compounds. Atest compound can be determined to be therapeutic by screening using thescreening methods of the present invention. A “known therapeuticcompound” refers to a therapeutic compound that has been shown (e.g.,through animal trials or prior experience with administration to humans)to be effective in such treatment or prevention.

[0039] The term “sample” as used herein is used in its broadest senseand includes environmental and biological samples. Environmental samplesinclude material from the environment such as soil and water. Biologicalsamples may be animal, including, human, fluid (e.g., blood, plasma andserum), solid (e.g., stool), tissue, liquid foods (e.g., milk), andsolid foods (e.g., vegetables).

DESCRIPTION OF THE INVENTION

[0040] Two major dendrimer compositions that are produced commerciallyusing a divergent synthetic strategy include: poly(amidoamine) or PAMAMdendrimers and poly(propyleneimine) or POPAM dendrimers. PAMAMdendrimers with ethylene diamine cores are produced by DendritechIncorporated of Midland, Mich. (Tomalia et al., Angew. Chem. Intl. Edit.29:138-175 [1990]; and Naylor et al., J. Am. Chem. Soc. 111:2339-2341[1989]), while POPAM dendrimers with diaminobutane cores are produced byDSM of Herleen, The Netherlands (De Brabander et al., Angew. Chem. Int.Ed. 32:1308 [1993]; and De Brabander et al, Macromol. Symp. 102:9[1996]). PAMAM dendrimers are remarkably biofriendly syntheticsubstances that have been incorporated into a considerable number ofexperimental diagnostic and therapeutic compositions. Unfortunately, theproduction of high generation PAMAM dendrimers, capable of encapsulatingguest molecules requires numerous synthetic rounds making thesecompounds costly and potentially less than monodisperse. POPAMdendrimers, in contrast, can be produced to higher dimensions with fewersynthetic rounds thus yielding a potentially more homogenous product ata reduced cost. The use of POPAM dendrimers, however, is typicallylimited to plastics, inks, adhesives, and catalysts, as POPAM dendrimersare fairly toxic to biological systems. Thus, new types of dendrimersare needed for various applications in the life sciences. In particular,new dendrimer compositions are desirable for drug delivery, medicalimaging, and gene transfection purposes.

[0041] To meet this need, the present invention provides noveldendrimeric copolymers composed of biocompatible PAMAM exteriors and lowcost POPAM interiors. These hybrid dendrimers (POPAM+PAMAM=POMAM) can besynthesized from a low generation POPAM dendrimer core according to areiterative process involving sequential Michael addition and amidation.Thus, the POMAM dendrimers of the present invention, which aresynthesized at a reduced cost as compared to similar sized PAMAMdendrimers, are expected to be biocompatible vehicles for drugs,contrast agents or nucleic acids.

[0042] I. POMAM Dendrimers

[0043] Briefly, the preparation of POMAM hybrid dendrimers involves adivergent synthesis consisting of two reiterating reactions. The firstreaction is a step growth process that involves Michael addition ofamino groups to the double bond of methyl acrylate (MA). The secondreaction is a chain growth process that involves amidation of theresulting terminal methyl ester with ethylenediamine (EDA). When POPAMdendrimers are used as the initiator cores, these syntheses may berepresented by the reactions in FIG. 2.

[0044] In the first step of this process, POPAM dendrimers of generation2, 3, or 4, with 16, 32, or 64 primary amine groups on their surfaces,were allowed to react under an inert nitrogen atmosphere with excess MAat room temperature for 24-48 hours, with the amount of time varyingproportionally with the number of primary amine groups on the startingmaterial. The resulting compounds are referred to as half generationPOMAM hybrid dendrimers. The second step of the process involvesreacting the newly-formed terminal esters with excess EDA to produce aPAMAM shell around the POPAM dendrimer. The amidation reactions wereperformed under inert nitrogen atmosphere in methanol at 2° C. and alsorequire 24-48 hours for completion. Thus, POMAM hybrid dendrimers of theinvention have twice as many primary amine groups on their surface asdid the starting material.

[0045]¹³C NMR spectroscopy is a sensitive measurement of the magneticenvironment of every carbon atom in a compound. The highly symmetricalPOPAM, PAMAM, and POMAM dendrimer structures places all of the terminalgroups in an almost equivalent magnetic environment, yielding verysimple spectra for molecules of such high molecular weights. Anydeviations from this symmetry, perhaps caused by errors in synthesis ordegradation of the material, are indicated by additional signals in the¹³C NMR spectrum (e.g., corresponding to the carbon atom in the vicinityof the structural defect). FIG. 3 provides a representative example of a¹³C NMR spectrum of a generation 2:1 POMAM dendrimer. The spectrumdepicted in FIG. 3 is essentially the superposition of the spectra ofPOPAM and PAMAM dendrimers. The peak at approximately 175 ppm clearlyindicates the PAMAM shell incorporation with the POPAM dendritic core.

[0046] As shown in FIG. 4, the theoretical and experimental molecularweights of the dendrimers are dependent upon the generation number ofthe POPAM core and the number of PAMAM shells added to the core.Experimental molecular weights were determined by GPC. Lower generationPOMAM hybrid dendrimers (2:1, 2:2, 3:1, 3:2, 4:1, and 4:2) exhibited avery narrow distribution in molecular weight indicating that thesedendrimers were relatively monodisperse. Higher generation POMAM hybriddendrimers (e.g., 2:3, 2:4, 3;3, 3;4, 4:3, and 4:4), in contrast,exhibited a narrow, bimodaldistribution in molecular weight. The second,higher molecular weight peak, accounted for less than 10% of the area ofthe first, lower molecular weight peak. The higher molecular weight peakmay correspond to that of aggregated dendrimers as the conditions usedfor GPC would be expected to allow aggregation to take place. That said,the aggregation behavior of the higher molecular weight hybrid dendrimerwas not observed on the HPLC eluogram. A representative HPLC eluogramfor a generation 2:2 POMAM dendrimer is shown in FIG. 5A, while FIG. 5Bis an HPLC eluogram showing a comparison of the generation 2:2 POMAMdendrimer with the PAMAM dendrimers of generations 0 through 7. FIG. 5Bclearly indicates that the hydrodynamic volume of the generation 2:2POMAM dendrimer lies between the hydrodynamic values of the generation 3and 4 PAMAM dendrimers.

[0047]FIG. 6 depicts the hydrodynamic diameter of the POMAM dendrimersas a function of the generation number of the core and the number ofshells covering the core. The relation is almost linear and reaches apeak value of approximately 83 angstroms.

[0048]FIG. 7A shows the HPLC eluograms of POMAM hybrid dendrimers ofgenerations 2:4, 3:4 and 4:4. The dendrimer preparations are relativelypure as evidenced by the appearance of a single narrow major peak. Onlya small amount of a higher molecular weight entity is present, which isin all likelihood due to aggregation of the lower molecular weightspecies. The inconsistent appearance of aggregates suggests that thisbehavior is highly influenced by the environment of the molecules. FIG.7B shows how the POMAM dendrimers asymptotically approach the liquidchromotographical properties of the PAMAM dendrimers.

[0049] The goal of obtaining high resolution AFM images of individualPOMAM hybrid dendrimers was challenging as dendrimer molecules possess ahigh concentration of surface functional groups, which often causes themto aggregate. High resolution images of individual PAMAM, POPAM, orPOMAM hybrid dendrimers of generation 5 or lower are difficult to obtainbecause of this problem (Jackson et al., Macromolecules 31:6259 [1998]).The larger dendrimers, however, and particularly the hybrid dendrimers,can be spread on a mica surface to show individual molecules inultra-dilute solutions. Spin-coating techniques are also helpful inpreparing uniform dendrimer films. FIG. 8 shows tapping mode AFM imagesof a generation 4:3 hybrid dendrimer. The globular particles which arerandomly deposited on the mica surface are substantially uniform in sizeindicating that they are essentially monodisperse. Although a fewaggregates are present in FIG. 8, most of the clusters represent singlemolecules.

[0050] It is well known that ionic strength, surface charge density, andconcentration or molecular environment are the important parameterscontrolling the degree of structural organization of the solution(Nisato et al., Macromolecules 32:5895-5900 [1999]). Addition of amonovalent salt such as potassium chloride or sodium chloride screensthe long range electrostatic interactions and leads to a gas-likestructure (e.g., disaggregated arrangement). Full generation dendrimershave a high density of primary amino groups on their surface. In thatcase, the surface charge densities of these molecules can be manipulatedby varying the pH of the solution, permitting the dendrimers to beviewed as nanoscopic polyelectrolyte particles.

[0051] Importantly, the POMAM dendrimers involve two sorts of aminegroups. The primary amine end groups (—NH₂) are on the dendrimerperiphery, and their number is equal to 2^((n+m+2)), with n representingthe generation of the POPAM core, and m representing the number of PAMAMshells. The tertiary amine groups (>N—) are situated at the branchingpoints in the molecule, and their number is equal to 2^((n+m+2))−2.These two types of amine groups if isolated, are characterized bydifferent pK values (Van Genderen et al., Polym. Mater. Sci. Eng. 73:336[1995]), which are negative logarithms of the acidic dissociationconstant for the protonated primary (pK^(pr)) and tertiary (pK^(t))amine groups correspondingly. The basicity of the primary amine group ishigher compared with that of the tertiary one (e.g., pK^(pr) is greaterthan pK^(t)).

[0052] The protonation behavior of the dendrimers was studied bypotentiometric titration. FIG. 9 shows a representative result obtainedfor a generation 2:2 POMAM dendrimer, titrated with 0.1 N HCl. Clearly,two sections can be distinguished, one for the primary amines at a highpH and one for the tertiary amines at a lower pH. The degree ofprotonation, which separates the regions was found to be equivalent tothe ratio between the primary and secondary amines. The back titration,done with 0.1 N NaOH, shows three sections representing three acidspresent in the solution. The first section corresponds to excess HCl,the second to the tertiary amine, and the third to the protonatedprimary amine, respectively. The primary amine groups on the surface ofthe dendrimer are the most basic amino moieties of the dendrimer with apK_(a) _(^(pr)) of ˜8.9. In contrast, the tertiary amines have a pK_(a)_(^(t)) of 5.6.

[0053] Thus, monodisperse POMAM hybrid dendrimers with POPAM cores andPAMAM shells were prepared using MA Michael addition and EDA amidationsteps under controlled conditions. The POPAM cores used were generation2, 3, and 4 dendrimers with 16, 32, and 63 primary amines, respectively.The observed 13C NMR, molecular weights, and hydrodynamic diameters wereclose to the theoretical values. Moreover, the AFM image indicates thatthe POMAM hybrid dendrimers were of a relatively uniform size.

[0054] II. Utilities

[0055] The following description provides exemplary utilities of thedendrimers of the present invention. These utilities find use with thePOMAM dendrimers of the present invention as well as other dendrimers(e.g., PAMAM dendrimers). The following applications are not intended tobe limited to the use of POMAM dendrimers.

[0056] A. Gene Transfection

[0057] Many previous studies have described methods to achievetransfection of cells in vitro and in vivo that employ the use of adevice to accelerate expression plasmid DNA (Lin et al., Int. J. Derm.39:161-170 [2000]; and Mahvi et al, Immunol. Cell Bio. 75:456-460[1997]). The expression plasmid DNA can be administered either alone(e.g., “naked” DNA) or coated onto the surface of metal particles suchas elemental gold or tungsten (Lai et al., DNA Cell Biol. 14:643-651[1995]), through the use of polycations (e.g., spermidine or PLG; SeeChen et al., J. Virol. 72:5757-5761 [1998]). There are inherent problemsassociated with both strategies.

[0058] “Naked” plasmid DNA is subject to structural damage byapplication of shear forces during acceleration and deceleration. Inaddition “naked” DNA is not a “solid” particle, it is a hydrodynamiccircular double helix polymer that can be easily deformed during bothacceleration and deceleration. As a result, “naked” DNA used as aballistic particle does not penetrate tissue and cell membranes well.

[0059] There are also inherent problems with the use of DNA bound tometal particles. Elemental metal particles are large in size and whenaccelerated develop significant kinetic energy that causes significantnonspecific trauma to tissues and cells. Because the process of bindingexpression plasmid DNA to the metal particles is indirect (e.g.,involves several steps of mixing small molecules with metal particles),there is no reliable method to adjust the stoichiometry of DNA moleculesbound per metal particle. Moreover, the manufacture of DNA coated heavymetal particles is not readily scaleable.

[0060] The use of biocompatible dendrimers (i.e., PAMAM or POMAMhybrids) using the methods of the present invention as carriers forballistic delivery of expression plasmid DNA obviates the problemsassociated with previously described systems. In fact PAMAM dendrimershave been successfully used as the DNA carriers for both in vitro and invivo transfections (Bielinska et al., Bioconjugate Chemistry 10:843-850[1999]; Bielinska et al., Biomaterials 21:877-887 [2000]; andKukowska-Latallo et al., Hum. Gene Ther. 11:1385-1395 [2000]). Thepresence of negatively charged amino groups on the surface of dendrimerswith PAMAM shells, allows for electrostatic interactions with variousforms of nucleic acids. In addition, PAMAM dendrimers arenon-immunogenic and biocompatible at the concentrations used in theformulations of dendrimer-DNA complexes described herein.

[0061] Experiments conducted during the development of the presentinvention demonstrate that utility of ballistric transfection usingdendrimers. There are numerous advantages conferred by the use of PAMAMterminated dendrimer/DNA complexes for ballistic transfection of cellsin vitro and in vivo. In the first place, PAMAM-terminated dendrimersprotect expression plasmid DNA from shear-induced damage duringacceleration and deceleration. Secondly, PAMAM-terminated dendrimersallow for manufacture of DNA complexes with specific and reproduciblestoichiometric ratios and surface charges (e.g., zeta potential). Theseproperties are important in determining the efficiency of ballisticmediated transfection, and the manufacture of the dendrimer/DNAparticles is readily scaleable. Thirdly, PAMAM terminated dendrimer-DNAcomplexes are of consistent size and mass and can be formulated as arelatively monodisperse suspension. Fourthly, because the mass of thePAMAM terminated dendrimer-DNA complexes is substantially less than thatof heavy metal particles, the kinetic energy of the complexes can befinely regulated in order to optimize cell membrane penetration andminimize nonspecific cell trauma. Lastly, because PAMAM-terminateddendrimers can be readily derivatized during manufacture, moietiesdesigned to alter mass or provide targeting or biochemical function canbe added to the polymers to optimize properties favorable for ballisticdelivery. (Balogh et al., J. Nanoparticle Research 1:353-369 [1999]).

[0062] In summary, the results obtained from using in situ and in vivomodel systems suggest that the use of dendrimer-DNA complexes forballistic transfection of cells is significantly more efficient than theuse of “naked” plasmid DNA. Specifically, greater transfectionefficiency is defined as a higher level of transgenic protein expressionfor a given dose of administered DNA. Equally important, dendrimer/DNAcomplexes obviate many of the inherent technical and manufacturingproblems associated with ballistic delivery of “naked” plasmid DNA orDNA indirectly bound to elemental heavy metal particles.

[0063] Examples 3-6 below provide a description of some preferredembodiments of the methods of the present invention.

[0064] B. Medical Imaging

[0065] In some embodiments of the present invention, dendrimers are usedfor medical imaging purposes by associating an imagable component withthe dendrimer. The present invention is not limited by the nature of theimaging component used. In some embodiments of the present invention,imaging modules comprise surface modifications of quantum dots (Seee.g., Chan and Nie, Science 281:2016 [1998]) such as zinc sulfide-cappedcadmium selenide coupled to biomolecules (Sooklal, Adv. Mater., 10:1083[1998]).

[0066] However, in preferred embodiments, the imaging module comprisesdendrimers produced according to the “nanocomposite” concept (Balogh etal., Proc. of ACS PMSE 77:118 [1997] and Balogh and Tomalia, J. Am. Che.Soc., 120:7355 [1998]). In these embodiments, dendrimers are produced byreactive encapsulation, where a reactant is preorganized by thedendrimer template and is then subsequently immobilized in/on thepolymer molecule by a second reactant. Size, shape, size distributionand surface functionality of these nanoparticles are determined andcontrolled by the dendritic macromolecules. These materials have thesolubility and compatibility of the host and have the optical orphysiological properties of the guest molecule (i.e., the molecule thatpermits imaging). While the dendrimer host may vary according to themedium, it is possible to load the dendrimer hosts with differentcompounds and at various guest concentration levels. Complexes andcomposites may involve the use of a variety of metals or other inorganicmaterials. The high electron density of these materials considerablysimplifies the imaging by electron microscopy and related scatteringtechniques. In addition, properties of inorganic atoms introduce new andmeasurable properties for imaging in either the presence or absence ofinterfering biological materials. In some embodiments of the presentinvention, encapsulation of gold, silver, cobalt, iron atoms/moleculesand/or organic dye molecules such as fluorescein are encapsulated intodendrimers for use as nanoscopi composite labels/tracers, although anymaterial that facilitates imaging or detection may be employed.

[0067] In some embodiments of the present invention, imaging is based onthe passive or active observation of local differences in density ofselected physical properties of the investigated complex matter. Thesedifferences may be due to a different shape (e.g., mass density detectedby atomic force microscopy), altered composition (e.g., radiopaquesdetected by X-ray), distinct light emission (e.g., fluorochromesdetected by spectrophotometry), different diffraction (e.g.,electron-beam detected by TEM), contrasted absorption (e.g., lightdetected by optical methods), or special radiation emission (e.g.,isotope methods), etc. Thus, quality and sensitivity of imaging dependon the property observed and on the technique used.

[0068] 1. Magnetic Resonance Imaging

[0069] Dendrimers havebeen employed as biomedical imaging agents,perhaps most notably for magnetic resonance imaging (MRI) contrastenhancement agents (See e.g., Wiener et al., Mag. Reson. Med. 31:1[1994]; an example using PAMAM dendrimers). These agents are typicallyconstructed by conjugating chelated paramagnetic ions, such asGd(III)-diethylenetriaminepentaacetic acid (Gd(III)-DTPA), towater-soluble dendrimers. Other paramagnetic ions that may be useful inthis context of the include, but are not limited to, gadolinium,manganese, copper, chromium, iron, cobalt, erbium, nickel, europium,technetium, indium, samarium, dysprosium, ruthenium, ytterbium, yttrium,and holmium ions and combinations thereof.

[0070] Dendrimeric MRI agents are particularly effective due to thepolyvalency, size and architecture of dendrimers, which results inmolecules with large proton relaxation enhancements, high molecularrelaxivity, and a high effective concentration of paramagnetic ions atthe target site. Dendrimeric gadolinium contrast agents have even beenused to differentiate between benign and malignant breast tumors usingdynamic MRI, based on how the vasculature for the latter type of tumorimages more densely (Adam et al., Ivest. Rad. 31:26 [1996]). Thus, MRIprovides a particularly useful imaging system of the present invention.

[0071] 2. Microscopic Imaging

[0072] The dendrimers of the present invention allow functionalmicroscopic imaging of tissues and provide improved methods for imaging.The methods find use in vivo, in vitro, and ex vivo. For example, in oneembodiment of the present invention, dendrimers of the present inventionare designed to emit light or other detectable signals upon exposure tolight. Although the labeled dendrimers may be physically smaller thanthe optical resolution limit of the microscopy technique, they becomeself-luminous objects when excited and are readily observable andmeasurable using optical techniques. In some embodiments of the presentinvention, sensing fluorescent biosensors in a microscope involves theuse of tunable excitation and emission filters and multiwavelengthsources (Farkas et al., SPEI 2678:200 [1997]). In embodiments where theimaging agents are present in deeper tissue, longer wavelengths in theNear-infrared (NIR) are used (See e.g., Lester et al., Cell Mol. Biol.44:29 [1998]). Dendrimeric biosensing in the Near-IR has beendemonstrated with dendrimeric biosensing antenna-like architectures(Shortreed et al., J. Phys. Chem., 101:6318 [1997]). Biosensors thatfind use with the present invention include, but are not limited to,fluorescent dyes and molecular beacons.

[0073] In some embodiments of the present invention, in vivo imaging isaccomplished using functional imaging techniques. Functional imaging isa complementary and potentially more powerful techniques as compared tostatic structural imaging. Functional imaging is best known for itsapplication at the macroscopic scale, with examples including functionalMagnetic Resonance Imaging (fMRI) and Positron Emission Tomography(PET). However, functional microscopic imaging may also be conducted andfind use in in vivo and ex vivo analysis of living tissue. Functionalmicroscopic imaging is an efficient combination of 3-D imaging, 3-Dspatial multispectral volumetric assignment, and temporal sampling: inshort a type of 3-D spectral microscopic movie loop. Interestingly,cells and tissues autofluoresce. When excited by several wavelengths,providing much of the basic 3-D structure needed to characterize severalcellular components (e.g., the nucleus) without specific labeling.Oblique light illumination is also useful to collect structuralinformation and is used routinely. As opposed to structural spectralmicroimaging, functional spectral microimaging may be used withbiosensors, which act to localize physiologic signals within the cell ortissue.

[0074] C. Therapeutic Agents

[0075] A wide range of therapeutic agents find use with the presentinvention. Any therapeutic agent that can be associated with a dendrimermay be delivered using the methods, systems, and compositions of thepresent invention.

[0076] Once inside the host cells or tissue, the biological agent hasbeen either directly or indirectly delivered to the target. In someembodiments, without limitation, direct delivery of the agent means thatthe agent, with or without the associated dendrimer, is secreted fromthe cell into the extracellular space, where it acts upon the targettissue or is taken up by the target tissue.

[0077] In some embodiments, without limitation, indirect delivery meansthat the biological agent is modified in the cell prior to beingsecreted. Modification can take place either while the agent is stillassociated with the dendrimer or after disassociation of the components.For example, the biological agent may be in an inactive form and isrendered active following the introduction of the dendrimer complex tohost cells or tissues. The biological agent, upon exposure to light or achange in pH (e.g., due to exposure to a particular intracellularenvironment), may be altered to assume its active form. Alternately, theagent may be attached to a protective linker (e.g., photo-cleavable,enzyme-cleavable, pH-cleavable) to make it inactive and become activeupon exposure to the appropriate activating agent, e.g., UV light, acleavage enzyme, or a change in pH. Indirect delivery may also comprise,in the case of transfection, the transcription of the nuclei acid toform a gene product, where the gene product is secreted to theextracellular space.

[0078] In other embodiments, the biological agent may not be secreted,but rather is retained within the cell where it may effect a change inthe biological activities of host cell, either directly or through aseries of signal transductions.

[0079] Degradation of the complex is useful because it eases thesecretion of the biological agent, or transcription if the biologicalagent is a nucleic acid. The dendrimer complexes tend to degrade in atime-dependent manner under physiological conditions. Other dendrimercomplexes resist degradation for a period of time under physiologicalconditions and then proceed to degrade. Degradation of the dendrimercomplexes may be influenced by the surface chemistries of the dendrimersutilized. For example, particular dendrimer complexes may be selected ordesigned that degrade under particular physiological conditions or underan exogenous cue, e.g., heat, light, ultrasonic energy, and the like,provided either at administration, or at a selected biological eventafter administration.

[0080] In addition, dendrimer complexes may comprise one or more layersof dendrimer structure with one or more biological agents associatedwith each layer. This may allow for the release over time of biologicalagents as the layers of dendrimer degrade. If the same biological agentwere used throughout the dendrimer, then a sustained release ofbiological agent would be obtained. Alternately, by using differingbiological agents, a sequential release of agents may be accomplished.Indeed, multiple dissimilar agents may be associated with each layer ofthe dendrimer.

EXAMPLES

[0081] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof. In the experimental disclosurebelow, the following abbreviations apply: MA (methyl acrylate); EDA(ethylene diamine); MeOH (methanol); DAB (1,4-diaminobutane); NMR(nuclear magnetic resonance); SEC (size exclusion chromatography); HPLC(high performance liquid chromatography); AFM (atomic force microscopy);GPC (gas phase chromatography); luciferase (Luc); chloramphenicolacethyltransferase (CAT); eq (equivalents); μ (micron); M (Molar); μM(micromolar); mM (millimolar); N (Normal); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams); μg (micrograms); ng (nanograms); L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nM (nanomolar); ° C. (degrees Centigrade); PBS (phosphatebuffered saline); hrs (hours); and RT (room temperature).

Example 1 POMAM Hybrid Dendrimer Syntheses

[0082] In this Example, the production of half and full generation POMAMhybrid dendrimers is described. Materials used for this purpose werepurchased from Aldrich and include: methanol (MeOH) with 99.93% purity,ether, methyl acrylate (MA), and ethylenediamine (EDA). The purity ofMeOH was 99.93%, while the purity of the remaining compounds was 99+%.EDA was distilled on a rotary evaporator at 2000 microns of mercury anda bath temperature of 36° C. The purified EDA was transferred to avessel and stored at −4° C. under a N₂ blanket. The Astromolpoly(propyleneimine) dendrimers of generations two through four (e.g.,DAB-dendr-(NH₂)₁₆ to DAB-dendr-(NH₂)₆₄) were obtained from either DSM orAldrich. Volumetric solutions of 0.1 M NaOH and 0.1 M HCl were alsopurchased from Aldrich, and used as received.

[0083] Generation 2:0.5, A: In a 50 ml three neck round bottom flaskequipped with a magnetic stirrer, pressure equalized dropping funnel andcondenser under a dry N₂ atmosphere, a solution of MA (6.41 ml,7.114×10⁻² mol) in 7.7 ml MeOH was cooled to 0° C. Then a solution of 3g (1.7785×10⁻³ mol) DAB-AM-16 polypropyleneimine hexadecaamine dendrimer(POPAM generation 2) in 10 ml MeOH (cooled to 0° C. under N₂) was addeddropwise. This mixture was stirred under N₂ at 36° C. for 48 hrs, andthe excess MA and MeOH was evaporated under a vacuum. To the residue, 3ml water was added, mixed carefully, and after freezing was lyophilizedto remove excess MeOH and MA, yielding a methyl-ester functionalizedPOPAM-core dendrimer, POMAM 2:0.5 (7.7 g, 97.5%).

[0084] Generation 2:0.5, B: To a mixture of MA (2.2495 g, 2.613×10⁻²mol) in 3 ml of MeOH cooled at 0° C. was added POPAM dendrimer(1,4-diaminobutane core, generation 2:0, with 16 NH₂ surface groups)(1.0203 g, 6.049×10⁻⁴ mol) in 3.5 ml of MeOH cooled at 0° C. Theresulting mixture was stirred at room temperature for 48 hrs. The MeOHand excess MA as volatiles were evaporated on a rotary evaporator at 34°C. and the resulting generation 2:0.5 dendrimer preparation was driedout at a vacuum of 500 microns of mercury to give 2.661 g (99.05%) ofthe title compound.

[0085] Generation 2:1: To a mixture of EDA (359.6 g, 5.9835 mol) in 100ml of MeOH cooled at 0° C. was added POMAM hybrid dendrimer, generation2:0.5 (1.0425 g, 2.347×10⁻⁴ mol) in 3 ml of MeOH cooled at 0° C. Thismixture was maintained at 0° C. for 48 hrs. After this reaction time themixture was warmed to room temperature. The volatiles were removed fromthe mixture on a rotary evaporator at 34° C. with a vacuum at 2000-500microns of mercury. The crude product was dissolved in MeOH and wasprecipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give1.246 g, 99.4% yield of the title compound.

[0086] Generation 2:1.5: To a mixture of MA (1.674 g, 194×10⁻² mol) in 2ml of MeOH cooled at 0° C. was added POMAM hybrid dendrimer (generation2:1, with 32 primary NH₂ surface groups; 1.2014 g, 2.25×10⁻⁴ mol) in 5ml of MeOH cooled to 0° C. The resulting mixture was stirred at roomtemperature for 48 hrs. The MeOH and excess MA as volatiles wereevaporated on a rotary evaporator at 34° C. and the generation 2:1.5dendrimer preparation was dried out under a vacuum of 500 microns ofmercury to give 2.404 g (98.5%) of the title compound.

[0087] Generation 2:2: To a mixture of EDA (1438.4 g, 23.934 mol) in 400ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer (generation2:1.5; 2.186 g, 2.015×10⁻⁴ mol) in 18 ml of MeOH cooled to 0° C. Thismixture was maintained at 0° C. for 72 hrs. After this reaction time themixture was warmed to room temperature. The volatiles were removed fromthe mixture on a rotary evaporator at 34° C. with a vacuum at 2000-500microns of mercury. The crude product was dissolved in MeOH and wasprecipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give2.472 g, 97.02% yield of the title compound.

[0088] Generation 2:2.5: To a mixture of MA (1.31 g, 1.52×10⁻² mol) in 2ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer (generation2:2, with 64 NH₂ surface groups; 1.002 g, 7.92×10⁻⁵ mol) in 5 ml of MeOHcooled to 0° C. The resulting mixture was stirred at room temperaturefor 72 hrs. The MeOH and excess of MA as volatiles were evaporated on arotary evaporator at 34° C. and G-2:2.5 was dried out at a vacuum of 500microns of mercury to give 1.804 g (96.2%) of the title compound.

[0089] Generation 2:3: To a mixture of EDA (2157.6 g, 35.901 mol) in 600ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer (generation2:2.5; 1.804 g, 7.62×10⁻⁵ mol) in 16 ml of MeOH cooled to 0° C. Thismixture was maintained at 0° C. for 72 hrs. After this reaction time themixture was warmed to room temperature. The volatiles were removed fromthe mixture on a rotary evaporator at 34° C. with a vacuum at 2000-500microns of mercury. The crude product was dissolved in MeOH and wasprecipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give1.6584 g, 79.8% yield of the title compound.

[0090] Generation 2:3.5: A mixture of MA (0.37 g, 4.3×10⁻³ mol) in 1 mlMeOH cooled to 0° C. was added to a heterophase POMAM dendrimer(generation 2:3, with 128 NH₂ surface groups; 0.3052 g, 1.12×10⁻⁵ mol)in 3 ml of MeOH cooled to 0° C. The resulting heterophase mixture wasstirred at room temperature for 96 hrs. The MeOH and excess of MA asvolatiles were evaporated on a rotary evaporator at 34° C. and thegeneration 2:3.5 dendrimer preparation was dried out under a vacuum of500 microns of mercury to give 0.4387 g, 79.5% yield of the titlecompound.

[0091] Generation 2:4: To a mixture of EDA (1078.8 g, 17.95 mol) in 300ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer (generation2:3.5; 0.4387 g, 8.9×10⁻⁶ mol) in 5 ml of MeOH cooled to 0° C. Thismixture was maintained at 0° C. for 120 hrs. After this reaction timethe mixture was warmed to room temperature. The volatiles were removedfrom the mixture on a rotary evaporator at 34° C. with a vacuum at2000-500 microns of mercury. The crude product was dissolved in MeOH andwas precipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give0.4963 g, 98.7% yield of the title compound.

[0092] Generation 3:0.5, A: In a 50 ml three neck round bottom flaskequipped with magnetic stirrer, pressure equalized dropping funnel andcondenser under dry N₂ atmosphere a solution of MA (5.41 ml, 6.008×10⁻²mol) in 6.5 ml MeOH was cooled to 0° C. Then a solution of 3 g(7.511×10⁻⁴ mol) DAB-Am-32 Polypropylenimine hexadecaamine dendrimer(generation 3:0) in 10 ml MeOH (also cooled to 0° C. under dry N₂) wasadded dropwise. This mixture was stirred under nitrogen at 34° C. for 48hrs, and the excess MA and MeOH was evaporated in a vacuum. To theresidue 3 ml of water was added, mixed carefully, and after freezing waslyophilized to remove excess MeOH and MA, yielding methyl-esterfunctionalized POPAM core dendrimer, POMAM 3:0.5, (7.02 g, 98.3%).

[0093] Generation 3:0.5, B: To a mixture of MA (2.2182 g, 2.577×10⁻²mol) in 3 ml of MeOH cooled at 0° C. was added POPAM dendrimer(1,4-diaminobutane core, generation 3:0, with 32 NH₂ surface groups;1.1912 g, 3.39×10⁻⁴ mol) in 3.5 ml of MeOH cooled to 0° C. The resultingmixture was stirred at room temperature for 48 hrs. The MeOH and excessMA as volatiles were evaporated on a rotary evaporator at 34° C. andgeneration 3:0.5 was dried out under a vacuum of 500 microns of mercuryto give 3.0858 g, ˜100% yield of the title compound.

[0094] Generation 3:1: To a mixture of EDA (719.2 g, 11.967 mol) in 200ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer (generation3:0.5; 1.0377 g, 1.15×10⁻⁴ mol) in 13 ml of MeOH cooled to 0° C. Thismixture was maintained at 0° C. for 48 hrs. After this reaction time themixture was warmed to room temperature. The volatiles were removed fromthe mixture on a rotary evaporator at 34° C. with a vacuum at 2000-500microns of mercury. The crude product was dissolved in MeOH andprecipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give1.233 g, ˜100% yield of the title compound.

[0095] Generation 3:1.5: To a mixture of MA (1.679 g, 1.95×10⁻² mol) in2 ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer(generation 3:1, with 128 NH₂ surface groups; 1.2728 g, 1.176×10⁻² mol)in 5 ml of MeOH cooled to 0° C. The resulting mixture was stirred atroom temperature for 48 hrs. The MeOH and excess of MA as volatiles wereevaporated on a rotary evaporator at 34° C. and the generation 3:1.5dendrimer preparation was dried out under a vacuum of 500 microns ofmercury to give 2.451 g, 95.4% of the title compound.

[0096] Generation 3:2: To a mixture of EDA (2157.6 g, 35.901 mol) in 600ml of MeOH cooled at 0° C. was added POMAM hybrid dendrimer (generation3:1.5; 2.323 g, 1.064×10⁻⁴ mol) in 18 ml of MeOH cooled to 0° C. Thismixture was maintained at 0° C. for 72 hrs. After this reaction time themixture was warmed to room temperature. The volatiles were removed fromthe mixture on a rotary evaporator at 34° C. with a vacuum at 2000-500microns of mercury. The crude product was dissolved in MeOH and wasprecipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give2.659 g, 98.3% yield of the title compound.

[0097] Generation 3:2.5: To a mixture of MA (1.615 g, 1.876×10⁻² mol) in2 ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer(generation 3:2, with 128 NH₂ surface groups; 1.242 g, 4.88×10⁻⁵ mol) in5 ml of MeOH cooled to 0° C. The resulting mixture was stirred at roomtemperature for 72 hrs. The MeOH and excess MA as volatiles wereevaporated on a rotary evaporator at 34° C. and generation 3:2.5 wasdried out under a vacuum of 500 microns of mercury to give 2.185 g,94.2% of the title compound.

[0098] Generation 3:3: To a mixture of EDA (5753.6 g, 95.736 mol) in1600 ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer(generation 3:2.5; 2.185 g, 4.56×10⁻⁵ mol) in 18 ml of MeOH cooled to 0°C. This mixture was maintained at 0° C. for 72 hrs. After this reactiontime the mixture was warmed to room temperature. The volatiles wereremoved from the mixture on a rotary evaporator at 34° C. with a vacuumat 2000-500 microns of mercury. The crude product was dissolved in MeOHand was precipitated out by addition of ether. This purification processwas repeated three times. The precipitate was dried very carefully togive 1.9742 g, 78.5% yield of the title compound.

[0099] Generation 3:3.5: A mixture of MA (0.37 g, 4.3×10⁻³ mol) in 1 mlMeOH cooled to 0° C. was added to a heterophase POMAM dendrimer(generation 3:3, with 256 NH₂ surface groups; 0.3022 g, 5.5×10⁻⁶ mol) in3 ml of MeOH cooled to 0° C. The resulting heterophase mixture wasstirred at room temperature for 96 hrs. The MeOH and excess MA asvolatiles were evaporated on a rotary evaporator at 34° C. and thegeneration 3:3.5 dendrimer preparation was dried out under a vacuum of500 microns of mercury to give 0.5088 g, 93.2% yield of the titlecompound.

[0100] Generation 3:4: To a mixture of EDA (2157.6 g, 35.9 mol) in 600ml MeOH cooled to 0° C. was added POMAM hybrid dendrimer (generation3:3.5; 0.5088 g, 5.2×10⁻⁶ mol) in 15 ml of MeOH cooled to 0° C. Thismixture was maintained at 0° C. for 120 hrs. After this reaction timethe mixture was warmed to room temperature. The volatiles were removedfrom the mixture on a rotary evaporator at 34° C. with a vacuum at2000-500 microns of mercury. The crude product was dissolved in MeOH andwas precipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give0.5736 g, 98.4% yield of the title compound.

[0101] Generation 4:0.5, A: In a 50 ml three neck round bottom flaskequipped with magnetic stirrer, pressure equalized dropping funnel andcondenser under dry N₂ atmosphere a solution of MA (5.02 ml, 5.575×10⁻²mol) in 6.1 ml MeOH was cooled to 0° C. Then a solution of 3 g(3.485×10⁻⁴ mol) DAB-Am-64 Polypropylenimine hexadecaamine Dendrimer(generation 4:0) in 10 ml MeOH (also cooled to 0° C. under dry N₂) wasadded dropwise. This mixture was stirred under N₂ at 36° C. for 48 hrs,and the excess MA and MeOH was evaporated in a vacuum. To the residue 3ml of water was added, mixed carefully, and after freezing waslyophilized to remove excess MeOH and MA, yielding methyl-esterfunctionalized POPAM-core dendrimer, POMAM 4:0.5, (6.83 g, 99.8%).

[0102] Generation 4:0.5, B: To a mixture of MA (1.7775 g, 2.065×10⁻²mol) in 2 ml of MeOH cooled to 0° C. was added POPAM dendrimer(1,4-diaminobutane core, generation 4:0, with 64 NH₂ surface groups;1.0287 g, 1.435×10⁻⁴ mol) in 3.5 ml of MeOH cooled to 0° C. Theresulting mixture was stirred at room temperature for 48 hrs. The MeOHand excess MA as volatiles were evaporated on a rotary evaporator at 34°C. and the generation 4:0.5 dendrimer preparation was dried out under avacuum of 500 microns of mercury to give 2.5112 g, 96.2% of the titlecompound.

[0103] Generation 4:1: To a mixture of EDA (1438.4 g, 23.934 mol) in 400ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer, (generation4:0.5; 1.0238 g, 5.63×10⁻⁵ mol) in 13 ml of MeOH cooled to 0° C. Thismixture was maintained at 0° C. for 48 hrs. After this reaction time themixture was warmed to room temperature. The volatiles were removed fromthe mixture on a rotary evaporator at 34° C. with a vacuum at 2000-500microns of mercury. The crude product was dissolved in MeOH and wasprecipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give1.21 g, ˜100% yield of the title compound.

[0104] Generation 4:1.5: To a mixture of MA, (1.503 g, 1.75×10⁻² mol) in2 ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer(generation 4:1, with 128 NH₂ surface groups; 1.1732 g, 5.39×10⁻⁵ mol)in 5 ml of MeOH cooled to 0° C. The resulting mixture was maintained andstirred at room temperature for 48 hrs. The MeOH and excess of MA, asvolatiles were evaporated on a rotary evaporator at 34° C. and thegeneration 4:1.5 dendrimer preparation was dried out under a vacuum of500 microns of mercury to give 2.15 g, 91.4% yield of the titlecompound.

[0105] Generation 4:2: To a mixture of EDA (3596 g, 59.835 mol) in 1000ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer (generation4:1.5; 1.865 g, 4.26×10⁻⁵ mol) in 18 ml of MeOH cooled to 0° C. Thismixture was maintained at 0° C. for 72 hrs. After this reaction time themixture was warmed to room temperature. The volatiles were removed fromthe mixture on a rotary evaporator at 34° C. with a vacuum at 2000-500microns of mercury. The crude product was dissolved in MeOH and wasprecipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give2.116 g, 97.4% yield of the title compound.

[0106] Generation 4:2.5: To a mixture of MA (1.32 g, 1.53×10⁻² mol) in 2ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer (generation4:2, with 256 NH₂ surface groups; 1.015 g, 1.99×10⁻⁵ mol) in 5 ml ofMeOH cooled to 0° C. The resulting mixture was stirred at roomtemperature for 72 hrs. The MeOH and excess MA as volatiles wereevaporated on a rotary evaporator at 34° C. and the generation 4:2.5dendrimer preparation was dried out under a vacuum of 500 microns ofmercury to give 1.427 g, 75.4% yield of the title compound.

[0107] Generation 4:3: To a mixture of EDA (7192.0 g, 119.67 mol) in2000 ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer(generation 4:2.5; 1.427 g, 1.48×10⁻⁵ mol) in 15 ml of MeOH cooled to 0°C. This mixture was maintained at 0° C. for 72 hrs. After this reactiontime the mixture was warmed to room temperature. The volatiles wereremoved from the mixture on a rotary evaporator at 34° C. with a vacuumat 2000-500 microns of mercury. The crude product was dissolved in MeOHand precipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give1.64 g, 99.8% yield of the title compound.

[0108] Generation 4:3.5: A mixture of MA (0.38 g, 4.4×10⁻³ mol) in 1 mlof MeOH cooled to 0° C. was added to a heterophase POMAM dendrimer(generation 4:3, with 512 NH₂ surface groups; 0.3107 g, 2.8×10⁻⁶ mol) in3 ml of MeOH cooled to 0° C. The resulting heterophase mixture wasstirred at room temperature for 96 hrs. The MeOH and excess MA asvolatiles were evaporated on a rotary evaporator at 34° C. and thegeneration 4:3.5 dendrimer preparation was dried out under a vacuum of500 microns of mercury to give 0.3922 g, 69.9% yield of the titlecompound.

[0109] Generation 4:4: To a mixture of EDA (2876.8 g, 47.9 mol) in 800ml of MeOH cooled to 0° C. was added POMAM hybrid dendrimer (generation4:3.5; 0.3922 g, 2.0×10⁻⁶ mol) in 3 ml of MeOH cooled to 0° C. Thismixture was maintained at 0° C. for 120 hrs. After this reaction timethe mixture was warmed to room temperature. The volatiles were removedfrom the mixture on a rotary evaporator at 34° C. with a vacuum at2000-500 microns of mercury. The crude product was dissolved in MeOH andprecipitated out by addition of ether. This purification process wasrepeated three times. The precipitate was dried very carefully to give0.4411 g, 98.2% yield of the title compound.

Example 2 POMAM Hybrid Dendrimer Characterization

[0110] In this Example, the techniques used to characterize the POMAMhybrid dendrimers of the present invention are described. The techniquesutilized for characterization include: NMR, SEC, HPLC, potentiometrictitration, and AFM.

[0111] For ¹H and ¹³C NMR measurements, a Bruker AVANCE DRX 500instrument was used. Approximately 30-40 mg/ml D₂O solutions was usedfor these investigations.

[0112] The SEC eluograms were obtained using an Alliance Waters 2690Separation Module combined with triple detectors: Waters 2487 DualAbsorbance Detector, Wyatt DAWN DSP Laster Photometer, and Optilab DSPInterferometric Refractometer at 30° C. The module was equipped with aTosoHaas TSK-GEL Guard PWH (06762), 7.5×7.5 cm, 12 μm (DS 1140), G 2000PW (05761), 10 μM (DS 1014), G 3000 PW (05762), 10 μM (DS 1016), and G4000 PW (05763), 17 μm (DS 1017) columns. A 0.1 N citric acid solution(pH 2.72 adjusted with sodium hydroxide, and containing 0.025% sodiumazide) was used as the mobile phase and for making sample solutions forSEC analysis. A nominal flow rate setting of 1.0 ml/min and an injectionvolume of 50 μl was used.

[0113] For HPLC measurements, a Beckman System Gold instrument was usedwhich was equipped with a solvent module (126) and a UV detector (166).A 0.1 M trifluoroacetic acid eluent was used with a flow rate of 1ml/min, in a C-18 reverse column at room temperature.

[0114] The potentiometric titration of dendrimers in aqueous solutionwas done using an ORION pH meter (model 230A) with and Oric glasscombined electrode (5107 BN) at room temperature. For samplepreparation, a 0.1 M NaCl solution was used, prepared from high purityNaCl (99.999%) and Milli-Q water (18 Mohm/cm).

[0115] For AFM measurements, samples on mica were examined using aTopoMetrix 2000 Discoverer instrument under ambient conditions.Ultrathin films of the POMAM hybrid dendrimers were prepared byspin-coating the dilute solutions on to freshly cleaved mica, which wasair-dried at room temperature. It was not possible to obtain stableimages using the contact mode, because the scanning tip appeared to movethe molecules. This problem was circumvented by using the tapping modefor imaging. Si probes having a spring constant of ca. 30 N/m were usedat a resonance frequency of ca. 200 to 300 kHz. A 7 μm scanner (x, y,and z directions) calibrated by TopoMetrix was used to collect the data.

Example 3 Preparation of Dendrimer-DNA Complexes and ParticleAcceleration

[0116] In this Example, the preparation of PAMAM-terminateddendrimer/DNA complexes and the ballistic transfer of the dendrimer/DNAcomplexes is described. It is important to note that the invention isnot necessarily specific to the method employed to achieve accelerationof the dendrimer/DNA complexes. An important component of the inventionis the use of dendrimer/DNA complexes of a specific kinetic energy atthe surface of the cells or tissues that are to be transfected.

Formulations of Dendrimer-DNA Complexes Used for Ballistic Transfection

[0117] Generation 5 EDA core PAMAM dendrimers were tested as aprototypic polymer, although the present invention is not limited to theuse of PAMAM dendrimers. In some experiments, generation 5 EDA corePAMAM dendrimers were modified during manufacture to contain smallamounts of silver (Ag) or gold (Au). Various formulations ofdendrimer/DNA complexes were prepared in water, 0.09% NaCl or in thepresence of modified -cyclodextrins. Size and population distribution ofthe dendrimer/DNA complexes was analyzed using a NICOMP Model 370particle sizer. The complexes were formed in water with plasmid DNA at aconcentration ranging from 0.05 mg/ml to 1 mg/ml and at the theoreticaldendrimer/DNA charge ratios of 0.0 1, 0.1 and 1. Dynamic Laser LightScattering (DLLS) analysis indicated that at the low charge ratios(e.g., 0.01 and 0.1) the mean dynamic diameter of the complex rangesfrom 5.3 to 59.7 nm depending upon the DNA concentration. However,complexes formed at the neutralizing charge ratio (e.g., 1.0) resultedin broadly polydispersed populations of particles containing theidentifiable fractions with a mean dynamic diameter of 459+/−29 nm (fora DNA concentration 0.05 mg/ml) and 909.4+/−66.7 nm (for a DNAconcentration of 0.1 mg/ml). The majority of particles existed in theform of large (>10 μm) aggregates and precipitates. The addition of theamphoteric -cyclodextrin at 0.05% or 0.1% (w/v) to dendrimer/DNAformulations resulted in the generation of almost monodispersed (>99%)particles with mean diameters of 5.3+/−0.5 nm and 17+/−11.4 nmrespectively and complete disappearance of the aggregates. Formulationsresulting in the most uniform distribution of dendrimer/DNA complexes ofthe 50 to 200 nm average particle size were used for the transfections.

[0118] During the development of the present invention, a commerciallyavailable hand held device was used to pneumatically accelerate thedendrimer/DNA complexes. The Biojector 2000 (Bioject) is a needle-freeinjection delivery system that utilizes compressed carbon dioxide as apower source for acceleration of materials in the form of aqueoussolutions or suspensions.

[0119] The Biojector can deliver dendrimer/DNA complexes in volumetricunit doses of 50 to 200 μl. The distance from the Biojector pneumaticorifice to the surface of the target can be regulated using spacers ofvarious lengths and diameters that can be attached to the Biojector.This orifice-target distance regulates the final kinetic energy of theparticles at the time of contact with skin or tissue (FIG. 10). In thesubsequent experiments, an orifice-target distance of 0 to 0.5 cm wasfound to be optimal for transfection of skin and mucosal cells.

[0120] All formulations tested contained a total of 100 μg plasmid DNAconsisting of equal amounts of pCF1Luc, pCF1CAT and pCF1 gal suspendedin a total volume of 100 μl water. Formulation I: DNA was complexed with50 μg of Ag nanocomposite generation PAMAM dendrimers at an ˜dendrimer/DNA charge ratio of 0.80. Formulation II: complexes wereenriched with 6.5 μg of the unmodified dendrimer which increased thecharge ratio to ˜0.9. Formulation III: 0.05% amphoteric -cyclodextrinwas added to Formulation II. Formulation IV: 0.05% sulphonated-cyclodextrin was added to formulation II.

Example 4 Ballistic Transfection of Human Skin with Dendrimer/DNAComplexes

[0121] Cadaveric split-thickness human skin was grafted onto the backsof SCID mice and used as a target for ballistic transfection. Allballistic transfections were done using a 1.5 cm adapter inserted intothe micro-orifice of the Biojector device. The skin was harvested 24 hrsafter ballistic transfection, as multiple 4 mm skin punch biopsies.Sonicated extracts of the punch biopsies were prepared and expression ofboth luciferase (Luc) and chloramphenicol acethyltransferase (CAT) wasdetermined using established methods.

[0122] Ballistic delivery of Formulations I and II resulted in similarlevels of luciferase expression (2.3×10⁵ RLU/mg and 3.3×10⁵ RLU/mg,respectively). CAT expression after transfection with Formulations I andII was found to be 84 and 47 pg/mg, respectively. Ballistic delivery ofFormulation III, in contrast, resulted in a 5 to 7 fold increase inluciferase activity reaching ˜1.7×10⁶ RLU/mg. Similarly, 120 pg/mg CATprotein was detected upon transfection with Formulation III. FormulationIV, containing sulphonated -cyclodextrin, was least effective andresulted in a 2 fold lower expression of both luciferase and CAT protein(FIG. 11).

[0123] The use of Ag or Au nanocomposite PAMAM dendrimers permitted thepharmacodistribution of the particles in skin following ballisticdelivery to be determined by transmission electron microscopy (TEM). Inparticular, through TEM the histologic and ultrastructural localizationof the particles was determined. In addition TEM was used to determinethe location of dendrimer-DNA complexes as a function of the kineticenergy at the surface of the skin. In the preliminary TEM analysis ofhuman skin grafts transfected with the nanocomposite PAMAM dendrimer/DNAcomplexes, irregular electron-dense deposits were detected in theexperimental but not in the control sections. The localization ofelectron dense particles in intracellular and intranuclear locations, aswell as, in the collagen-rich acellular matrix of the dermis wasconsistent with successful ballistic delivery of the dendrimer/DNAcomplexes (FIG. 12).

Example 5 Ballistic Transfection of Murine Skin with Dendrimer/DNAComplexes

[0124] Dose dependence and efficacy of ballistic transfection usingdendrimer/DNA complexes was tested in BALB/C mice. The shaved skin onthe dorsal side of animals was transfected with 10 or 50 μg doses ofpCF1CAT DNA alone or complexed with a PAMAM dendrimer. The dendrimer/DNAcomplexes at a charge ratio of 0.01 were prepared in 100 ml of water(e.g., DNA concentration of 0.5 and 0.1 mg/ml). All gene deliveries wereperformed without a spacer corresponding to an orifice-target distanceequal to 0. Ballistic transfection using dendrimer/DNA complexes wasfound to be more efficient than “naked” DNA alone at DNA doses of 10 and50 μg. The increase in transfection efficiency was most pronounced withdecreasing doses of DNA, approximately 2 fold for 50 μg DNA and 13 foldfor 10 μg DNA (FIG. 13). Additional ballistic transfections were doneusing a volumetric dose of 75 μl containing 10 or 50 μg “naked” DNA orDNA complexed to a generation 5 PAMAM dendrimer at a charge ratio of0.05 and an orifice-target distance of 0.5 cm. Two days followingballistic transfection using 10 μg of DNA in the dendrimer/DNA complex,the level of transgene expression observed was similar to that obtainedwith 50 μg of “naked” DNA, and more than 80 fold higher than with 10 μgof “naked” DNA (FIG. 5). Thus, ballistic transfection usingdendrimer-DNA complexes minimizes the total dose of DNA required toachieve a given level of transgene expression in vivo.

[0125] In addition, the use of dendrimer/DNA complexes greatly prolongedthe duration of the transgene expression in vivo. Murine skin wasballistically transfected using 10 or 50 μg of “naked” DNA or anequivalent dose of DNA complexed to a generation 5 PAMAM dendrimer.Seven days following transfection the levels of CAT protein detected inthe samples treated with dendrimer/DNA complexes were 15-20% of the peakvalues, while samples treated with “naked” DNA were at the lower limitsof assay detection (FIG. 14).

Example 6 Ballistic Transfection of Engineered Grafts with Dendrimer/DNAComplexes

[0126] The ballistic transfection of primary human oral mucosalkeratinocytes, and primary dermal keratinocytes present within a tissueengineered material has also been accomplished during development of thepresent invention. The tissue engineered material (Alloderm) used forthese experiments is suitable for intra-oral or dermal grafting (Izumiet al., J. Dental Res. 79:798-805 [2000]). Ballistic transfections ofprimary cell cultures of human fibroblasts and human oral keratinocytesgrown on the surface of Alloderm were performed with generation 5 PAMAMdendrimers complexed to pCF1CAT, pCF1Luc, or pCMVhVEGF121. ThepCMVhVEGF121 plasmid encodes a soluble form of vascular endothelialgrowth factor, an angiogenic protein that may have therapeutic value inthe context of promoting wound repair and healing. Regardless of thecell type present within the tissue engineered Alloderm, efficienttransfection was observed only in samples treated with PAMAMdendrimer-DNA complexes.

[0127] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention, which are obvious tothose skilled in relevant fields, are intended to be within the scope ofthe following claims.

We claim:
 1. A composition comprising a hybrid dendrimer having apoly(propyleneimine) interior and a poly(amidoamine) exterior.
 2. Thecomposition of claim 1, wherein said poly(propyleneimine) interior is adendrimer selected from the group consisting of a generation 2 dendrimerwith sixteen amine surface groups, a generation 3 dendrimer with 32amine surface groups, and a generation 4 dendrimer with 64 amine surfacegroups.
 3. The composition of claim 1, wherein said poly(amidoamine)exterior comprises one or more shells.
 4. The composition of claim 1,wherein said hybrid dendrimer has a 1,4-diaminobutane core.
 5. Thecomposition of claim 1, further comprising a guest molecule.
 6. Thecomposition of claim 5, wherein said guest molecule comprises a nucleicacid molecule.
 7. The composition of claim 5, wherein said guestmolecule comprises a metal.
 8. The composition of claim 5, wherein saidguest molecule comprises a drug.
 9. The method for preparing a hybriddendrimer comprising: a) providing an amine-terminatedpoly(propyleneimine) dendrimer, methyl acrylate, and ethylenediamine; b)reacting said amine-terminated poly(propyleneimine) dendrimer with saidmethyl acrylate to produce an ester-terminated compound; and c) reactingsaid ester-terminated compound with ethylenediamine to produce saidhybrid dendrimer.
 10. The method of claim 9, further comprising the stepof d) attaching a guest molecule to said hybrid dendrimer.
 11. Themethod of claim 9, wherein said amine-terminated poly(propyleneimine)dendrimer comprises a guest molecule.
 12. The method of claim 9, whereinsaid reacting steps are conducted in a methanol solvent under an intertnitrogen atmosphere.
 13. A composition comprising the hybrid dendrimerprepared according to the method of claim
 9. 14. The composition ofclaim 13, wherein said hybrid dendrimer has a hydrodynamic diameter offrom 10 to 100 angstroms.
 15. A method for transfecting cellscomprising: a) providing dendrimer/nucleic acid complexes and targetcells; and b) ballistically accelerating said dendrimer/nucleic acidcomplexes at said target cells under conditions such that nucleic acidenters said target cells.
 16. The method of claim 15, wherein saiddendrimer/nucleic acid complexes comprise PAMAM dendrimers.
 17. Themethod of claim 15, wherein said dendrimer/nucleic acid complexescomprise hybrid dendrimers having a poly(propyleneimine) interior and apoly(amidoamine) exterior.
 18. The method of claim 15, wherein saiddendrimer/nucleic acid complexes comprise metal particles.
 19. Themethod of claim 18, wherein said metal particles are selected from thegroup consisting of gold and silver particles.
 20. The method of claim15, wherein said dendrimer/nucleic acid complexes have a charge ratio of1 or less.
 21. The method of claim 15, wherein said dendrimer/nucleicacid complexes comprise 99% or greater monodispersed particles.
 22. Themethod of claim 15, wherein said ballistically accelerating step iscarried out by a ballistic device.
 23. The method of claim 22, whereinsaid ballistic device is held less than one centimeter from said targetcells during said ballistically accelerating step.